UA96231C2 - Generation of triacylglycerols from gums - Google Patents

Abstract

A method is disclosed for the generation of triacylglycerols from gums that have been separated from an oil product. The gums are treated with an enzyme having PLC activity, which results in the formation of diacylglycerols and phosphates, and treated with an enzyme having PLA activity, which results in the formation of lyso-phospholipids and free fatty acids. The diacylglycerols and the free fatty acids from these two separate reactions then combine in the presence of the enzymes to generate new triacylglycerol molecules.

Description

This application claims priority over US application Me11/970270 filed on Jan. 7, 2008 entitled "PREPARATION OF TRIACYLGLYCEROLS FROM CHEMES" in the name of SpgizIorpeg I.Oauyup.

This invention relates to the method of obtaining triacylglycerols from gums, which are recovered in the process of oil refining. More specifically, the present invention relates to the enzymatic process of processing various phospholipids and lecithins (commonly known as "gums") from vegetable oils to obtain or "generate" triacylglycerols (triglycerides or oils). The invention described herein is a further development of the inventions described in US patent application Me11/668921, filed January 30, 2007, and US patent application Me11/853339, filed September 11, 2007, which belong to the common applicant. and introduced in this text as a link.

Crude vegetable oils obtained by pressing or solvent extraction are a complex mixture of triacylglycerols, phospholipids, sterols, tocopherols, free fatty acids, trace metals and other minor compounds. Removal of phospholipids, free fatty acids and trace metals is necessary to obtain high-quality salad oil with a light color and mild taste for long-term storage. In the prior art, similar removal of phospholipids known as "gums" has been accomplished using various techniques, including aqueous degumming, acid degumming, alkaline degumming, and enzymatic degumming.

Most of these degumming methods are characterized by significant losses of oil along with separated gums.

The aforementioned patent applications describe methods of removing phospholipids from oil compositions by co-treatment of oil compositions with the enzymes RI A (phospholipase A) and RI C (phospholipase C). Treatment with two enzymes can be sequential or simultaneous. Unexpectedly, the kinetics of enzymatic reactions were found to be accelerated to a greater extent than expected when two enzymes were used together than when either enzyme was used alone. In addition, it was found that the reactions proceed faster than expected when the enzymes are used together, even if the reaction conditions were not selected for at least one of the enzymes. It was also found that when enzymes are used together, the reaction can proceed in less than about 1 hour, and can even proceed in 30 minutes.

It is expected that as a result of the processing of oil compositions with the enzymes RHA and RI C, certain side products of the reaction will be obtained, which must be removed from the processed oil. These co-products include phosphate-containing residues cleaved from phospholipids by RiC enzymes, free fatty acids cleaved from phospholipids by RHA enzymes, and lyso-phospholipids obtained as a result of cleaving a free fatty acid from a phospholipid.

Lyso-phospholipids and any phosphate-containing by-products must be removed from the treated oil composition, and the other by-products listed above are expected to be removed along with the lyso-phospholipids in the heavy fraction known as "gums".

U.S. Patent 5,061,498 relates to a process for the conversion of fats and oils, which involves treating fats and oils containing unsaturated glycerides with lipases of two or more types differing in specificity for fatty acids and/or specificity for the position of esterification in the presence of small amounts of water, to obtain fats and oils with a low content of incomplete glycerides. In the disclosed embodiment, lipase is used

P, as it will carry out the reaction in any of the three positions of the glycerol skeleton. A target fatty acid, such as oleic acid, can be added to a composition containing unsaturated glycerides in the presence of a lipase specific for the target fatty acid, such as lipase P. The presence of lipase E favors the preferred fatty acid reaction over by other fatty acids that may be present, and the presence of lipase P promotes the esterification of the predominant fatty acid at any position on the backbone of incomplete glycerides. The concentration of water is mostly less than 1500 m.h. (parts per million), more specifically from 10 to 200 ppm.

The object of this invention is to provide a method of processing the separated gums to obtain edible oil products that would otherwise be wasted.

In addition to the work described in the two aforementioned patent applications, studies were conducted on gums that were separated from oils treated with RI A/RI C. The gums were expected to contain free fatty acids and diacylglycerols present in amounts proportional to the amount of phospholipids, that were present in the original oil composition, which were subjected to the action of enzymes. Instead, the amount of free fatty acids and diacylglycerols was significantly lower than expected theoretically. Based on such an unexpected result, it was concluded that free fatty acids and diacylglycerols, which were by-products of the reactions of phospholipids with RIA and RI C, respectively, reacted with each other in the presence of RIA and RI C enzymes, with the formation of useful triacylglycerols, thus , in fact, generating new oil molecules that did not exist before the RI A/RI C treatment process began. Thanks to this, it was discovered that a combination of RIA and RIS enzymes can be used to process separated phospholipids, regardless of the method of separation of these phospholipids, to generate new molecules of triacylglycerols.

Accordingly, the present invention relates to a method of generating triacylglycerols from oil gums, a method comprising (a) providing an oil composition containing a certain amount of oil gums, wherein said gums contain phospholipids, (B) separating said oil gums from said oil composition to obtain a first fraction that is substantially free of oil gums and a second fraction containing said separated oil gums, (c) treating said second fraction with one or more enzymes with RI A activity to produce free fatty acids, and (4) treating said second fraction by one or more enzymes with PI C activity for the formation of diacylglycerols, in such a way that the indicated fatty acids and the indicated diacylglycerols interact with each other in the presence of at least one of the indicated enzymes, forming triacylglycerols.

In fig. 1 shows the structural formulas of phospholipid and triacylglycerol.

In fig. 2 shows the three stereospecific positions of the phospholipid.

In fig. The structures of four common functional groups that can be attached to the phosphate group of a phospholipid are given.

In fig. 4 shows four different sites of enzymatic action in a phospholipid molecule.

In fig. 5 shows the reaction of phospholipid in the presence of the RHA enzyme and water, which leads to the formation of lysophospholipid and fatty acid.

In fig. 6 shows the reaction of phospholipid in the presence of the enzyme RI C and water, which leads to the formation of diacylglycerol and phosphate.

In fig. 7 shows the structural formula of phosphatidylcholine.

In fig. 8 shows the structural formula of phosphocholine.

Almost all losses associated with the refining process of vegetable oils occur during the removal of phospholipids.

As shown in fig. 1, phospholipids contain a phosphate group at one of the two ends of the glycerol backbone, while triacylglycerol contains three fatty acids. To distinguish derivatives, a system of stereospecific numbering ("Zp") is used. In fig. 2 shows the three stereospecific positions of the phospholipid.

The phosphate group of a phospholipid is "hydrophilic," or "water-loving," meaning that both the phosphate and the X functional group are attracted to water. The fatty acid chains of phospholipid NO and A2 are "lipophilic," or "lipophilic," meaning that they are attracted to lipids. Since the phospholipid molecule has both a hydrophilic functional group and lipophilic fatty acid chains, it is an excellent natural emulsifier.

The emulsifying properties of phospholipids will lead to the removal of two molecules of phospholipid and one molecule of triacylglycerol when removing phospholipids from vegetable oils.

Phosphate-containing functional group of phospholipid, marked in fig. 1 as "X", determines the degree of hydrophilicity.

Functional group X in fig. 1 may be a group of any of several different known types, a small portion of which is shown in FIG. 3.

Phospholipids containing choline and ethanolamine as functional groups have the highest affinity for water, while acids, acid salts (calcium, magnesium, and iron salts) and inositol have a much lower affinity for water. Phosphatidic acid and phosphatidic acid salts are commonly known as "non-hydrated phospholipids" or phospholipids.

The phospholipid content of an oil is usually defined as "phosphorus content" in parts per million. Table 1 describes the usual amounts of phospholipids present in most vegetable oil crops and the distribution of the various functional groups, in percentages, of the phospholipids in the oils Table 2 describes the usual distribution of phospholipids present in lecithin (soybean gum). In Table 2, "as is" means the normal phospholipid composition separated from vegetable oil with concentrated oil (2 molecules of phospholipid and 1 molecule of oil), which gives an acetone-insoluble content of 67 95. "Normalized" means the phospholipid composition without impurity of oil, which gives an acetone-insoluble content of 100 95. Table C describes the molecular weights of the major types of phospholipids, lyso-phospholipids, and corresponding non-lipid phosphorus compounds. The term "lyso-phospholipid", when used in Table C and throughout this application, means a phospholipid from which one of the fatty acid groups was cleaved by lipase.

The molecular weight of oleic acid is 282.48, and the molecular weight of diacylglycerol, in which the fatty acid is oleic acid (C18:1), is 620.99.

Table 1

Distribution and amount of phospholipids in oils from common oil crops 11111111 Soyeva oil | Canola oil Sunflower oil

Table 2

Normal distribution and amount of phospholipids in soybean gums 11111111 | Percentage "| Percentage" Normalized "(phosphatidiphserin .////////77777111111111117117117111111111111111111111111111111111C1C (phosphatidylovic acid,/-/-|/77777777

Table C

Molecular weight of common phospholipids and shen compounds nen | wine | Six

Molecular weight Molecular weight Molecular weight

(Silic acid81 | 7777171717171711721907777777711 71111111 45742 | yuyu g sh FG

Phospholipids can be partially or completely removed from vegetable oils using several different technologies; the most common are aqueous degumming, acid degumming, alkaline refining and enzymatic degumming. The present invention, which describes the preparation of oils from gums, may be applied to gums obtained by any of these processes; for illustrative purposes, enzymatic degumming will be explained in detail.

Enzymatic degumming, also known as "enzymatic refining", is used when the goal is to completely remove phospholipids from the oil. Basically, according to the prior art, enzymatic degumming is applied to oils that have previously been degummed by some other method, usually aqueous degumming. For use as a food additive, enzymatically degummed oil can then be subjected to lightening and deodorization, a technology known in the industry as "physical refining."

Enzymatic degumming provides better oil yield than aqueous, acid or alkaline degumming, with improved economic performance.

The enzymatic reaction changes the nature of the phospholipid, cleaving off various functional groups of the molecule.

Functional groups and breakdown products can mainly be considered as "fatty substances" and "phosphorus-containing substances". The enzymatic reaction reduces the emulsifying properties of the obtained phospholipids in such a way that when separating gum from oil, losses are reduced and oil is preserved. Enzymes that show activity in relation to phospholipids are usually called "phospholipases". The classification of phospholipases is based on the position in the phospholipid molecule with which the enzyme reacts, and the known types of phospholipases are RI AT, RI Ag, RIS and RIU.

The positions in the phospholipid molecule with which different types of phospholipases interact are shown in fig. 4. Phospholipase

B is an additional enzyme known in the art. This enzyme cleaves the last fatty acid in position 5p-1 or 5p-2 (Fig. 2) of lyso-phospholipid. Brief information about various phospholipases and their products and the reactions they carry out are presented in Table 4.

Table 4 11111100 Fatty substances.food/// | /////// Phosphorus-containing substances./7/://З

Each type of phospholipase has its own reaction rate and requires its own optimal reaction conditions in terms of pH, water content, and temperature. RI A used by itself requires a reaction time of at least 4 hours, while RI C used by itself requires a reaction time of about one hour. It is known that the enzymatic treatment should take place at a pH of less than or equal to 8, to reduce unwanted saponification of the oil, but RI A has an optimal reaction pH of 4.5, while RI C has an optimal reaction pH of 7.0.

Also, each of the enzymes has different heat resistance. RIA enzymes are denatured at approximately 50 "C, and RI C enzymes are denatured at approximately 65 "C.

Amino acid sequences with phospholipase activity are widely reported in the literature and described in patents, and some of them are known to have activity against phospholipids present in vegetable oils. All this is known in the art.

One of RI AT's commercial products with phospholipase enzymatic activity is the firm's phospholipase AT

Momogutez (I esyavze? ha). As described in the instructions for use of Momogutev "Ov 5. Bayiv" Me 2002-185255-01 and 2002-05894-03, this product can be mixed with degummed oil in 1-1.5 95 aqueous citrate-Maon buffer at 4.5" pHe7.0 and 40 "C"eTe55 "b. Under the specified conditions, RI A1 selectively hydrolyzes the fatty acid in the position opposite the phosphate functional group on the glycerol backbone, and yields polar lyso-phospholipids and polar fatty acids. As shown in fig. 4, the phospholipid molecule loses one hydrophobic functional group, that is, a fatty acid, leaving a lyso-phospholipid that now carries a hydrophilic phosphate group and a hydrophilic alcohol group.

Having two hydrophilic sites, the lyso-phospholipid molecule becomes water-soluble and loses its emulsifying properties. Thus, when the water phase is separated from the oil phase, the lyso-phospholipid is removed with the water phase and does not capture the oil, while the fatty acid molecule separated from the phospholipid remains in the oil. In the techniques of the known prior art, this fatty acid molecule must be removed in the subsequent deodorization procedure. Thus, the method of degumming with the help of RI AT reduces losses during refining, since no neutral oils are removed together with lyso-phospholipids in the aqueous phase, so that the only component that is removed is the unwanted lyso-phospholipid obtained from the original phospholipid molecule .

The theoretical amount of fatty acids that can be produced by the interaction of the gums with an RHIA-type enzyme can be calculated by determining the total amount of phospholipids in the gums, the amount of each type of phospholipid, and finally the change in molecular weight that occurs when the phospholipid is converted to lysophospholipid. , for each type of phospholipid present. The percentage of phospholipid can be calculated by multiplying the amount of elemental phosphorus, measured in parts per million, by 31 (the molecular weight of phosphorus is 30.97) and dividing by 10,000. The amounts of each type of phospholipid can be calculated by multiplying the total number of gums by the normal distribution of the phospholipid of each type known for a specific type of oil. Finally, the amount of fatty acid released can be determined based on each type of phospholipid.

For example, for unrefined soybean oil containing 800 m.ch. of phosphorus with a "normalized" phospholipid distribution (Table 2), assuming that the fatty acids attached to the phospholipids are oleic acid (C18:1), the expected amount of fatty acids released can be calculated as follows:

First, the total content of the phospholipid present is calculated.

The total amount of phospholipids - (800 m.h./1000000)x31x100-2.48 Vol.

Then the amount of phospholipid of each type is calculated.

Phosphatidylcholine - (2.48x47.21)/100-1.17 95

Phosphatidylethanolamine - (2.48x19.92)/100-0.49 95

Phosphatidylserine - (2.48x0.56)/100-0.01 Fo

Phosphatidylinositol « (2.48х23.40)/100-0.58 90

Phosphatidic acid - (2.48х8.91)/100-0.22 Fo

Finally, the amount of fatty acids released by the reaction of each type of phospholipid in the gums with RI A is determined by multiplying the amount of each type of phospholipid by the percentage of free fatty acid (EEA), the percentage of fatty acid being calculated as the residue after removing the amount of lyso-phospholipid (see . table 3), as follows:

ERA with RO - 1.17x(1-(521.67/786.15))-0.39 95

ERA with RE: 0.49x(1-(479.52/744.00))-0.18 95

ERA with RB - 0.01x(1-(522.56/787.03))-0.00 Fo

ERA with RI -: 0.58x(1-(599.50/863.98))-0.18 90

ERA with RA - (0.22x(1-(457.22/721.90)-0.08 95

The expected total amount of free fatty acids formed is 0.83 bo

Although enzymatic degumming offers significant advantages for oil refineries, it also has certain disadvantages. One disadvantage is that the reaction of the enzyme with phospholipids can be slow and take a long time. In particular, the reaction of phospholipase A enzymes with phospholipids can take several hours, depending on such reaction parameters as pH, temperature, relative concentrations and mixing conditions. Such long reaction times can significantly negatively affect the total economic benefit of the enzymatic degumming technology. Due to the slowness of the RA reaction, enzymatic degumming is usually performed on oil compositions that have previously undergone aqueous degumming. Thus, to obtain a product with a sufficiently low phosphorus content for the intended purpose, the oil can undergo degumming twice.

It is known in the art that RI C enzymes interact with phospholipid by selective hydrolysis of the phosphate functional group, as shown in fig. 6. At the output of the reaction, diacylglycerol ("CAC") and a phosphatidyl group are obtained. The diacylglycerol molecule no longer carries a phosphate functional group and does not need to be removed from the oil. For example, the reaction of Phosphatidylcholine (ROS), fig. 7, with R/C leads to the formation of RA and the phosphate functional group shown in fig. 8, more commonly known as phosphocholine or "C". The method of degumming with the participation of RI C reduces oil losses in the refining process by retaining the oil-soluble RAS and simultaneously removing the water-soluble phosphate functional group. In the process of removing the aqueous phase, no neutral oils are lost, since the phospholipid has been destroyed. However, the RI C enzyme does not interact with any of the lipids present in the oil. Usually, PI C does not interact with phosphatidic acid (PA) or phosphatidylinositol (PI), shown in fig. 3, although RI-specific RIS, called RiI-RI C, are known. In addition, both RA and RI are non-hydrated phospholipids that remain in the oil after aqueous degumming. Therefore, oil treated only with the RI C enzyme must undergo additional treatment with alkali or other enzymes to remove the remaining gums.

The theoretical amount of diacylglycerols produced by the reaction of gums with an enzyme of the RI C type can be calculated by determining the percentage of phospholipids in the oil, the amount of each type of phospholipid in the oil of a particular species, and finally, the change in molecular weight that occurs when the phospholipid is converted to OD for of phospholipids of each type present in crude oil. The percentage of phospholipid in the oil can be calculated by multiplying the amount of elemental phosphorus, measured in parts per million, by 31 (the molecular weight of phosphorus is 30.97) and dividing by 10,000. The number of individual phospholipids can be calculated by multiplying the total amount of gum by the normal distribution of each type of phospholipid . Finally, the amount of diacylglycerol can be determined as the amount of each type of phospholipid reaction product.

For example, for unrefined soybean oil containing 800 m.ch. of phosphorus with a "normalized" phospholipid distribution (Table 2), assuming that the fatty acids attached to the phospholipids are oleic acid (C18:1), the expected amount of diacylglycerols released can be calculated as follows:

First, the percentage of each type of phospholipid is calculated as described above.

Then, the percentage of diacylglycerols (AC) of each type released by the reaction of RI C with the gums can be determined by multiplying the amount of phospholipid of each type by the percentage of diacylglycerols (Table 3), the amount of DAC is calculated as the residue after removing the amount of phosphate groups, as follows: pAa with PO - 1.17x(1-(165.10/786.15))-0.93 95 pAa with RE - (0.49x(1-(123.10/744.00)-0.41 95 pAa with RB5 - (0.01х(1-(166.08/787.03)-0.01 Fo rda with RI) - (0.58х(1-(243.00/863.98)-0.42 90 pA with RA - (0.22x(1-(100.92/721.90)-0.19 95

The total amount of diacylglycerols obtained is 1.96 95

This invention relates to the enzymatic processing of phospholipids and phosphorus-containing oil compositions in order to obtain new triacylglycerol molecules. The authors of the invention unexpectedly discovered that the use of a combination of phospholipases with RIA and RIS activity not only leads to the cleavage of specific "groups", but also recombines the fatty acid (REA) separated in the reaction with RIA and diacylglycerol (DAC) obtained as a result of the reaction with RIS. with the output of triglyceride or oil. In particular, phospholipase A (RIA) interacts with a phospholipid molecule to form BA and lyso-lecithin, while Phospholipase C (PIC) interacts with another phospholipid molecule to form JAS and phospholecithin. The fatty acid obtained in the reaction with RI A and the CAC obtained as a result of the reaction with RI C are then combined by esterification in the presence of one or more enzymes to obtain a new molecule of triacylglycerol (TAC).

The present invention is particularly useful for application in the further processing of gums that have been separated from crude oil by such methods as aqueous refining, acid refining or alkaline refining, or enzymatic refining, except for refining with the joint use of RI A and RI C enzymes. It is believed that will preferably bring the pH of the alkali-refined gums to 8 or less before proceeding with the oil preparation steps of the present invention.

Oils that can be processed in accordance with the present invention may include, but are not limited to, the following oils: canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hemp oil, linseed oil, mango stone, meadowfoam oil, safflower oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, canola oil, rice bran oil, safflower oil, camellia oil (Sateiia zazapdca), soybean oil, sunflower oil, tallow oil, camellia oil (Sateia yg2ybaki) and vegetable oil, and any combination of the above oils.

The phospholipase A enzyme used according to the method of this invention can be both the phospholipase AT enzyme and the phospholipase A2 enzyme. The phospholipase C enzyme used in the present invention can be a phospholipase C enzyme and/or an inositol-specific phospholipase C. Many enzymes of the phospholipase A and phospholipase C families are commercially available; and it is anticipated that such enzymes and their equivalents will prove suitable for use in accordance with the present invention.

According to the method of the invention, the different phospholipases used together in the enzymatic degumming method of the present invention can be mixed with each other before being added to the oil being processed.

Alternatively, they can be added to the oil separately, either sequentially or simultaneously.

The degumming method of this invention is carried out at a pH below about 8, preferably between about 3 and 7, and most preferably between about 4 and 5. The pH value of the enzymatic degumming reaction can be achieved by adding known buffers. It is well known that citric acid and sodium hydroxide are suitable for this purpose. If necessary, other buffer substances can be used to adjust the pH under specific reaction conditions.

According to the present invention, the temperature of the enzymatic degumming process can be in the range of approximately 40-80 "C, preferably in the range of approximately 40-60 "C, and more preferably in the range of approximately 45-"C. It was unexpectedly found that with the method of this degumming of the RGA invention can work at a temperature that exceeds the enzyme's own optimum of 45 "C and is close to the optimal operating temperature

RI C, without excessive denaturation.

After the process of forming the oil in the gums is complete and the newly formed oil is separated from the gums, the newly formed oil may undergo such further processing steps as are known in the art, such as clarification or deodorization, which may be necessary or desirable depending on the end use for which the re-formed oil is the oil formed is intended.

Various preferred embodiments of the invention are set forth below in the examples, along with control examples, using the conditions of the known state of the art. In each of the examples listed below, the top-drive stirrer was a NeidoIrp model EIIesiog Ka stirrer with flat blades; was used at 90 rpm for normal mixing and 350 rpm for intensive mixing. A sedimentation centrifuge Oe I ama! was used for continuous separation. Sugo-Tevieg with a "rotor". The rotor of the centrifuge was closed with an installed screw plug. Shear mixing was performed using a Shiga-Tytakh 50-45 homogenizer with a rotor-stator 2450 at 10,000 rpm. The RI A1 enzyme was used

And esyaze? Sha (batch number I ММО5007) supplied by Momogutevs A/5 (Denmark). The enzyme RI S Rygipe"m was used (RIS batch number З?0В0002А1 or 9080004А1), supplied by the Megepit company

Sogrogayop (Zap Oiedo, Saiogpia). The amount of phospholipids remaining in the processed oil was measured in m.h.

P according to the method approved by the American Society of Petrochemists, Са 20-99, "Analysis of phosphorus content in oil using optical emission spectroscopy with inductively coupled plasma". Free fatty acid (FAA) was measured by the CA 5Ba-40 method approved by the American Society of Petrochemists. Moisture content was measured by the Ca 20-25 method approved by the American Society of Petrochemists. Neutral oil was determined by the method described in the appendix below. Acetone-insoluble material, including phospholipid, was determined by method da 4-46, approved by the American Society of Petrochemists. Acidity was determined by the 6-55 method approved by the American Society of Petrochemists. The methods of measuring P-31 (phosphorus-31) NMR and diacylglycerol (AC) by high-performance liquid chromatography with an evaporative light scattering detector (НРИ С-ЕИИ 50) were carried out following the protocols provided by Megepit Corporation (hereinafter known as Oimegza Corporation), "Analytical Profiling of Laboratory of Phospholipase C-Mediated Degumming of Vegetable Oils," at the 2007 American Society of Petrochemists Conference.

Among the following examples, Examples 1-10 are directly related to Examples 13, 14, 18, 23, 24, 27, 29, 31, 33, and 36 of the aforementioned US patent application Me11/853339, filed on Sep. 11, 2007, except that values of free fatty acids (EPA) and diacylglycerols present in enzymatically degummed oil were measured by the methods outlined above and included in this document.

Example 1 1999.1 g of raw soybean oil containing 769.5 m.h. of phosphorus compounds was heated to 75-80 with normal stirring using a top-drive stirrer. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred (shear deformation) for 1 min. The oil was subjected to normal mixing for 1 hour using a top-drive stirrer. The oil was cooled with stirring at a normal speed to a temperature of 40 "C, then 2.4 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 5.0. At a temperature of 40 "C, which is maintained, 1.5008 g of Megepisht Rigipe"m (RIS lipase, batch number 908Ш0002А1) was added, followed by the addition of 30 g of deionized water, and the entire mixture was stirred for 120 seconds. The oil mixture was stirred at normal speed for 60 minutes. At a temperature of 40 "C, which supported, added 0.2132 g of I esyaze? Opga firm

Momogutevs (RIA1 lipase, batch number I ММО5007) and the whole mixture were stirred for 120 s. The oil mixture was stirred at a normal speed for 60 min at a temperature of 40 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. The proportion of residual phosphorus-containing substances in the oil sequentially degummed with RI C and then RI AT was 6.5 m.h., the share of ERA was 0.56 95 and AC 0.69 9.

Example 2 2010.5 g of crude soybean oil containing 785.1 m.h. of phosphorus compounds were cooled to 60 "C with normal stirring using a top-drive stirrer. At a temperature of 60 "C, which is maintained, 1.5316 g of Megepisht's Rigiipe (RI C lipase, batch number 9080002A1) and 0.2073 g were added

Yessyaze? ShPyga of the company Momogutev5 (RIA7 lipase, batch number YIMMO5007) with the subsequent addition of 30 g of deionized water and the whole mixture was stirred for 45 seconds. The oil mixture was stirred at normal speed for 60 min at a temperature of 60 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. The proportion of residual phosphorus-containing substances in the degummed oil, co-treated with a mixture of РІС and РГА1 enzymes at neutral pH, was 109.6 m.h. The share of ERA was 0.61 95 and SAC 0.74 9.

Example From 2005.3 g of unprocessed soybean oil containing 742.9 m.h. of phosphorus compounds was heated to 75-80 with normal stirring using a top-drive stirrer. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred for 1 min. The oil was subjected to normal stirring for 71 hours using an overhead stirrer. The oil was cooled with stirring at a normal speed to a temperature of 60 "C, then 2.4 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for s. Citric acid and alkali formed a weak buffer with a pH of 5.0. At a temperature of 60 "C, that is supported, 0.7491 g of Rigiyipe "M of the Megepisht company (RI C lipase, batch number З0ОВО002А1) was added, followed by the addition of 60 g of deionized water, and the entire mixture was stirred for 45 s. The oil mixture was stirred at normal speed for 60 min. At a temperature of 60 "With what is supported, 0.1220 g of I esyaze was added? Opga firm

Momogutevs (RI Ai lipase, batch number I MMO5007) and the whole mixture were stirred for 45 seconds. The oil mixture was stirred at normal speed for 60 min at a temperature of 60 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. The proportion of residual phosphorus-containing substances in the oil sequentially treated with RIS, then RIA1, was 2.2 m. h. The share of EEA was equal to 0.58 95 and OD 0.42 95.

Example 4 2002.0 g of crude soybean oil containing 747.3 m.h. of phosphorus compounds was heated to 75-80 with normal stirring using a top-drive stirrer. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred for 1 min. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was cooled with stirring at a normal speed to a temperature of 60 "C, then 1.8 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. At a temperature of 60 "C , which is supported, 2.2194 g of Rigiyipe "M of the Megepisht company (RI C lipase, batch number З0ОВО002А1) were added, followed by the addition of 60 g of deionized water, and the entire mixture was stirred for 120 s. The oil mixture was stirred at normal speed for 15 min. At the temperature 0.2198 g of I esiazeb ha firm was added to 60 "C, which is maintained

Momogutevs (RIA1 lipase, batch number I ММО5007) and the whole mixture were stirred for 120 s. The oil mixture was stirred at normal speed for 15 min at a temperature of 60 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. The proportion of residual phosphorus-containing substances in the oil sequentially degummed by RI C and RI AT was 4.6 m .h. The share of ERA was 0.37 95 and TO 0.42 or.

Example 5 2000.8 g of raw soybean oil containing 747.3 m.h. of phosphorus compounds was heated to 75-80 with normal stirring using a top-drive stirrer. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred for 1 min. The oil was subjected to normal stirring for 71 hours using an overhead stirrer. The oil was cooled with stirring at a normal speed to a temperature of 50 "C, then 1.8 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. At a temperature of 50 "C , which is supported, added 2.2500 g of Rigypem of the Megepit company (RI C lipase, batch number З?0ВО002АТ) and 0.2216 g of I esiaze?

Momogutez (RI A1 lipase, batch number GMO5007), followed by the addition of 90 g of deionized water and the entire mixture was stirred for 45 seconds. The oil mixture was stirred at normal speed for 120 min at a temperature of 50 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. The proportion of residual phosphorus-containing substances in the oil jointly treated with the enzymes РИС and РИГ A was 1.8 m .h. The share of ERA was 0.67 95 and TO 0.40 95.

Example 6 2010.0 g of crude soybean oil containing 810.8 m.h. of phosphorus compounds were cooled to 50 C with normal stirring using a top-drive stirrer. At a temperature of 50°C, which is maintained, 2.2608 g of Rigipe "mM of the Megepisht company (RI C lipase, batch number З0ВО002А1) was added, followed by the addition of 30 g of deionized water, and the entire mixture was stirred for 60 seconds. The oil mixture was stirred at normal speed for 60 min. At a temperature of 50 "C, which is maintained, 0.1172 g of I esiaze was added?

Shyga of the company Momogutez (RI A1 lipase, batch number I ММО5007) and the entire mixture was stirred for 60 seconds. The oil mixture was stirred at normal speed for 60 min at a temperature of 50 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. The proportion of residual phosphorus-containing substances in the oil sequentially degummed with RI C and RI A! at neutral pH was 72.6 m.h.

The share of ERA was 0.53 95 and CAC 1.03 95.

Example 7 2006.3 g of crude soybean oil containing 795.3 m.h. of phosphorus compounds was heated to 75-80 with normal stirring using a top-drive stirrer. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred for 1 min. The oil was subjected to normal stirring for 1 hour using an overhead stirrer. The oil was cooled with stirring at normal speed to a temperature of 50 "С, then 2.4 ml of a 4-molar solution of sodium hydroxide was added and the mixture was stirred for s. Citric acid and alkali formed a weak buffer with a pH of 5.0. At a temperature of 50 "С, which is supported, 1.5373 g of Rippe"m of the Megepit company (RI S lipase, batch number З?0ВО002АТ) and 0.1168 g of I esiaze? of the company were added

Momogutez (RI A1 lipase, batch number ГМО5007) with the subsequent addition of 90 g of deionized water and the entire mixture was stirred for 120 seconds. The oil mixture was stirred at normal speed for 30 min at a temperature of 50 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. The proportion of residual phosphorus-containing substances in the oil, jointly treated with the enzymes РИС and RI AT at pH 5.0, was 1.9 m.h. The share of ERA was 0.17 95 and ODO 0.42 9B.

Example 8 2003.6 g of unprocessed soybean oil containing 784.8 m.h. of phosphorus compounds was heated to 75-80 with normal stirring using a top-drive stirrer. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred for 1 min. The oil was subjected to normal stirring for 71 hours using an overhead stirrer. The oil was cooled with stirring at normal speed to a temperature of 40 "C, then 1.8 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. At a temperature of 40 "C , which is supported, added 1.4603 g of Rigiyipe "m of the Megepit firm (RI C lipase, batch number 9080002A1) and 0.1021 g of I esyaze? Opga firm

Momogutez (RI A1 lipase, batch number I МО5007) with the subsequent addition of 40 g of deionized water and the entire mixture was stirred for 120 seconds. The oil mixture was stirred at normal speed for 120 min at a temperature of 40 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. was 10.7 ppm. The share of ERA was equal to 0.48 95 and TO 0.83 95.

Example 9 2000.4 g of unprocessed soybean oil containing 697.7 m.h. of phosphorus compounds was heated to 75-80 with normal stirring using a top-drive stirrer. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred for 1 min. The oil was subjected to normal stirring for 71 hours using an overhead stirrer. The oil was cooled with stirring at normal speed to a temperature of 40 "C, then 1.8 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. At a temperature of 40 "C , which is supported, 1.508 g of Ripipe"m of the Megepitz company (RIS lipase, batch number 9980002A1) and 0.1022 g of I esyaze? Opga company were added

Momogutez (RI A1 lipase, batch number ГМО5007) with the subsequent addition of 90 g of deionized water and the entire mixture was stirred for 120 seconds. The oil mixture was stirred at normal speed for 30 min at a temperature of 40 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. The proportion of residual phosphorus-containing substances in the oil degummed together with the enzymes RI C and RI AT at pH 4.5 , was 2.2 m.h. The share of ERA was 0.20 95 and ODO 0.41 9B.

Example 10 1999 g of raw soybean oil containing 695.1 m.h. of phosphorus compounds was heated to 75-80 with normal stirring using a top-drive stirrer. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred for 1 min. The oil was subjected to normal mixing using a top-drive stirrer for 1 hour. The oil was cooled with stirring at a normal speed to a temperature of 60 C, then 1.8 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for 10 seconds. Citric acid and alkali formed a weak buffer with a pH of 4.5. At a temperature of 60 "С, which is maintained, 1.5296 g of Rigip"m of the Megepisht company (RI C lipase, batch number 9080002A1) and 0.1241 g were added

Yessyaze? ShPyga of the company Momogutev5 (RIA7 lipase, batch number YIMMO5007) with the subsequent addition of 90 g of deionized water and the entire mixture was stirred for 120 seconds. The oil mixture was stirred at normal speed for 30 min at a temperature of 60 "C. Then the enzyme-treated oil was centrifuged and the fractions of the separated oil and raw gums were collected separately. The proportion of residual phosphorus-containing substances in the oil degummed together by the enzymes РИС and RIGA1 at pH 4.5 was 5.2 m.h. The share of ERA turned out to be equal to 0.36 95 and DS 0.44 vol.

The inventions described in the patent applications cited above were focused on the development of a method of enzymatic degumming, where the reaction conditions are selected to yield the smallest possible amounts of residual phosphorus with the smallest amounts of technological additives, equipment and time requirements. Upon completion of the research and all subsequent analytical tests, it was surprisingly found that the amount of fatty acids and diacylglycerols formed in the oils did not correspond to the theoretical amount that should be obtained. Based on the fact that PO and PE interacted with RI C, it is expected that the content of ODOi will increase by approximately 1.16-1.35 95, depending on the content of the original phospholipids. Since the RI AT enzyme interacts with all phospholipids, the EPA content is expected to increase by approximately 0.77-0.83 95, again depending on the content of the starting phospholipids. In addition, if RIS interacts with all ROS and PE, then the expected increase in EBA content due to the interaction of the remaining phospholipids with RI A will be approximately 0.53-0.59 96 for the examples described above. The greatest increase in the content of UASi was 0.63 95 and fatty acid 0.12 (example 6); both values are lower than expected.

Analysis of the separated heavy phase, or "gums", was performed in Examples 1-10 above to determine whether the gums were hydrated and physically removed; what is the length of the reaction of each enzyme and whether the reactions of one enzyme prevail over the reactions of another. Table 5 summarizes the phospholipid composition/distribution analysis performed for the separated heavy phase by P-31 NMR methods, showing the amount of unreacted phospholipid, the amount of lyso-phospholipid, and the amount of free phosphate, where all amounts are expressed as percent by weight of the sample. (Note: No phospholipid data for Example 6 due to microbial contamination of the sample).

Table 5 Ave. ROS | RE | RI | RA | SRS | RE | RI | SRA | "C" | "Ye Y" | A 1 | 000 | 000 | 173 | 000 | 181 | 022 | 571 | 7.01 | 3.82 | 275 | 0.00 | 081 2 | 000 | 000 | ol | 0.00 | 1.43 | 227 | bli | 422 | 456 | 3.40 | 0.00 | 0.82

With | 702 | 0.00 | 2.77 | 0.00 | 9.55 | 0.21 | 8.30 a.m 13.205| 2.47 | 1.34 | 0.00 | 0.50 4 | 099 0.00 | 476 | 0.95 | 5.28 | 0.32 | 593 | 10.81 3.73 | 1.81 | 0.00 0.49 | 1.62 | 0.00 | 3.35 | 0.78 | 1229) 021 | 8.28 | 11.91) 1.49 | 089 | 0.00 | 046 6|- | - 1-1 - 1-11 - 1-11 11-11 7 | 000 | 016 | ate | 0.00 | i.32 | 0.38 | 5.35 | 1.24 | 3.56 | 2.95 | 0.00 | 0.93 8 | 000 | 015 | 1.08 | 0.00 | i.60 | 0.67 | 6.37 | 1.98 | 4.37 | 3.57 | 0.00 | 133 9 | 008 000 1 3.03 | 0.00 | 1.93 | 0.08 | 5.07 | 10.80 | 3.85 | 2.58 | 0.00 0.84 (lo | 2.09 | 0.00 | 426 | 0.00 | 722 | 0.24 | 6.90 | 12.81| 2.06 | 114 | 0.00 | 0.49

Example 1 describes the sequential addition of enzymes at pH 5, which allows ROS and PE to first interact with the enzyme RI C and then allows the remaining RI, RA, RS and PE to interact with the enzyme RI A1. The RI C enzyme was in contact with the oil for 60 min at 40 "C before the RI A enzyme was added, thus allowing the enzyme to interact with all the RS and RE present in the oil without competing with the RI A enzyme. After the initial treatment

The RIA enzyme was added to the RIS, so that the RIA could hydrolyze the phospholipids remaining in the oil. The content of the remaining phosphorus compounds, which was noticed to be higher, was successfully reduced to 6.5 ppm. The starting oil contained 0.40 95 Aa and 0.41 95 EPA, compared to the obtained oil, where the content of ODS was 0.69 95 and PEA 0.56 95. If both enzymes interacted with specific phospholipids, then the content of OADSC should have increased by 1.29 to 1.69 95 and the content of EEA should have increased by 0.55 to 0.96 95. It turned out that in fact the content of SDS increases only by 0.29 95 and BA by 0.1595. The analysis of the gums by the P-31 NMR method showed that all the original phospholipids, except RI, were hydrolyzed. Significant amounts of lyso-RI and lyso-RA, and small amounts of lyso-RS and lyso-RE were present. Phosphorus compounds "C", "E" were also present in the reconstituted gums, as well as phosphorus compounds "A", which was surprising since it was published that RiC does not interact with RA. In addition, phosphoinositol was not detected. When comparing the distributions in the gums and the original oil, it turned out that 1.095 RAS and 0.40 95 BA are lost.

Example 2 describes a one-time addition of enzymes at pH 7 with 1.5 95 water and 45-second stirring.

Both enzymes were in contact with the oil for 60 min at 60 "С, thus, the enzymes competed with each other. The centrifuged oil contained residual phosphorus compounds of 109.6 ppm; the content of ODS increased by 0.34 9 and

EEA by 0.20 95. Analysis of the collected gums by the P-31 NMR method showed the presence of only small amounts of RI and the absence of RS, RE or RA. Therefore, all the original phospholipids reacted. Significant amounts of lyso- and phosphorus-containing compounds were detected in the gums, with the exception of "I". It was an unexpected discovery that the PAS content slightly increased compared to example 1, and the amounts of "C" and "E" were higher than in example 1.

Example C describes the sequential addition of enzymes at pH 5 with C 95 water and mixing for 45 s after the addition of each enzyme at 60 °C. The RI C enzyme was contacted with oil for 60 min before the RIA enzyme was added, and both enzymes had the opportunity to react for an additional 60 min.

Centrifuged oil contained 2.2 ppm. phosphorus compounds; the content of ODOx practically did not increase (from 0.40 to 0.42 95), and the content of EPA increased only by 0.17 95. If we compare the gums collected in example 3 with those from example 2, there was a strong increase in the content of lyso- compounds, while the content of all phosphorus compounds decreased. The data indicate that under these conditions reactions involving RI A predominate over reactions with RI C, even if RI C was added first.

The amount of UDS and EPA should have been much more than was present in the processed oil, based on the digestibility of the original phospholipids and the production of reaction products.

Example 4 describes the sequential addition of enzymes at pH 4,5,3 95 water and stirring for 120 s after the addition of each enzyme at 60 "C. The enzyme RI C was in contact with the oil for 15 min before the enzyme RI A was added, and both the enzymes were allowed to react for an additional 15 min. The centrifuged oil contained only 4.6 ppm of phosphorus compounds, the OAS content increased by only 0.02 905, while the EPA decreased by 0.04 95.

Analysis of the collected gums showed that some RS, RA and RI were not hydrolyzed. The content of lyso-compounds and phosphorus compounds was not as high as in example C, but still increased, taking into account the limitations in the interaction with enzymes.

Example 5 describes a one-time addition of enzymes at pH 4.5 with 4.595 water and 45-second stirring. Both enzymes were in contact with the oil for 120 min at 50 "C, so the enzymes competed with each other throughout the reaction. The centrifuged oil contained 1.8 ppm of residual phosphorus compounds; the RDA content did not increase at all, and the EBA content increased by 0 ,26 95. In the studied gums, the number of lyso compounds increased significantly and the number of phosphorus compounds decreased compared to the previous 4 examples, indicating the predominance of RI A reactions over RI C reactions in the reaction mixture.

Example 6 describes the sequential addition of enzymes at pH 7. The RI C enzyme was in contact with the oil for 60 min at "C before the RI A enzyme was added, so the enzyme had the opportunity to interact with all the PO and PE present in the oil without competition with the enzyme RI A. After the initial reaction with RI C, RI A was added so that RI A could hydrolyze the phospholipids remaining in the oil. The content of residual phosphorus compounds, as stated earlier, only decreased to 72.6 ppm. Starting oil contained 0.40 95 ODA and 0.41 95 EPA, while the treated oil contained 1.03 95 pAC and 0.53 95 REBRA.If both enzymes interacted with specific phospholipids, the content of OASC should have increased by 1.36 95 to 1.76 95, and the REA should have increased by 0.57 906 to a total content of 0.98.

In fact, the content of SAD increased by 0.63 905, and ERA only by 0.12 95. Analysis of gums by the P-31 NMR method was impossible due to microbial contamination of the sample.

Example 7 describes a one-time addition of enzymes. The enzymes were in contact with the oil sample for a total of 30 minutes at pH 5 of 4.5 95 water and at a temperature of 50 "C. The proportion of residual phosphorus-containing substances in the oil was only 1.9 ppm. The amount of all lyso-compounds decreased, especially lyso-RS. The number of phosphorus compounds was twice as high as in the analysis of the gum in example 5, indicating that under these conditions, the reactions of RI C prevail over the reactions of RI A. However, the content of OAE: practically did not increase (from 0.40 to 0.41 95) compared to the parent oil, and the EEA content did not increase to the expected total of 0.98 95, but actually decreased by 0.24 95 to a total EEA content of 0.17 Or.

Example 8 describes a one-time addition of enzymes at pH 4.5 with 295 water and 120-second stirring. Both enzymes were in contact with the oil for 120 min at 40 "С, therefore, the enzymes competed with each other during the entire reaction. The centrifuged oil contained 13.3 ppm of residual phosphorus compounds; the content of ODO increased by 0.43, and EPA by 0. 07 95. The content of lyso compounds decreased, while the content of phosphorus compounds was still higher than in example 7.

In example 9, a reaction similar to the reaction in example 8 is described, but instead of 2 90 water, 4.5 95 water was added to the reaction with the simultaneous action of enzymes, and the time of enzymatic exposure was 30 min instead of 120 min. The proportion of residual phosphorus-containing substances in the oil was 2.2 ppm. The amount of ODO in the oil remained practically the same as in the original oil, while the content of EPA decreased by 0.21 95. The low content of residual phosphorus compounds and the increased amount of all lyso-compounds, especially lyso-RA, indicate a high enzymatic activity of PI A. Based on this, it was expected that a very large amount of EPA should be formed during the reaction. The content of phosphorus compounds slightly decreased compared to Example 8, but the OAE content should have been significantly higher, according to the amounts of phosphorus compounds in the gums.

Example 10 was carried out with the simultaneous addition of enzymes at pH 4.5 with 4.5 95 water and stirring for 120 seconds. The enzymes were in contact with the oil for 30 minutes at 60 "C. The proportion of residual phosphorus-containing substances in the oil was 5.2 ppm. The amount of SAC in the oil remained almost the same as it was in the original oil (change from 0.40 to 0.44) , while the EPA content decreased by 0.05 95, while the amount of byproducts remained approximately the same as in the gums in Example 3. The reaction conditions in Examples 3 and 10 were different, but the yield according to the indicators of the remaining phosphorus compounds , CAC and ERA turned out to be approximately the same, indicating that the reaction is very stable in terms of TA formation.

Below, in Table 6, the initial amounts of phosphorus compounds, CAC and EPA in the starting oils for each of the above-described examples 1-10 are summarized, the theoretical amount of SDS and EPA that should be present in the treated oil if all the phospholipids in the starting oil were subjected to enzymatic action, and were obtained as a result of measurements of the amount of phosphorus compounds, CA and EPA in the processed oil.

Table 6 вне 00 еевюютевт Ivairnya linnosty in oosnennya processed oil (m.ch) со со со со (m.ch) о о 1 | 76565 | 040 | 041 | 769 | 096 | 65 | 069 | 056 ( 6 | 8108 | 040 | 041 | 776 | 098 | 726 | 103 | 053 8 1 7839 | 040 | 041 | 171 | 097 | 133 | 083 | 048 9 1 697.7 | 040 | 041 | 7157 | 091 | 22 | od | 020 lo | vom | 040 | 041 | 756 | 090 | 52 | 044 | 036 x The theoretical amount of ODSC includes newly formed from phosphatidylcholine and phosphatidylethanolamine. "x The theoretical amount of ERA includes newly formed from phosphatidylserine, phosphatidylinositol and phosphatidic acid.

Analysis of the gums by the P-31 NMR method confirmed that the gums were not hydrated and physically separated from the oil, as in "normal" processes of aqueous and/or acid degumming, but were hydrolyzed by the enzymes РИС and RIA.

The analysis confirmed the formation of split phosphorus compounds and the formation of lyso-lecithins (table 5). However, this does not explain why the levels of CAC and EPA were reduced in the treated oils. No information can be found in the prior art to describe why significant amounts of ODS and ERA are lost.

U.S. Patent Application Me11/668921 and U.S. Patent Application Me11/853339, owned by Rauyup et al., describe an enzymatic method for removing various phospholipids from vegetable oil to obtain degummed (degummed) oil using a combination of enzymes, according to which the reaction time is one hour or less.

The authors of the invention discovered a synergistic effect of RI C and RI A enzymes, improving the kinetics of the reaction from 2-6 hours when using enzymes individually to one or even less hours when using two enzymes together.

This invention is based on an unexpectedly low level of OAS and EPA content, which was revealed by further analyzes of oils treated in this way. Based on these data, the invention described in this document consists in the discovery that RI A and RI C apparently interact synergistically on a lipid matrix containing phospholipids and their accompanying products of hydrolysis by RIA/RIS enzymes to form triacylglycerols. Without intending to be bound by a particular theory, it is believed that cleaved diacylglycerol, a product of hydrolysis by the enzyme RIS, and cleaved fatty acids, a product of hydrolysis by the enzyme RHA, combine under the conditions of the enzymatic degumming reaction to form new triacylglycerols. It is theorized that the convergence or orientation, or both, of the two enzymes enables the formation of triacylglycerols during the release of diacylglycerols and fatty acids in the aqueous phase of the reaction.

An additional series of experiments was conducted with gums obtained from crude soybean oil by the traditional aqueous degumming method previously described in the prior art. Raw gums were obtained directly by industrial water degumming technology. Raw gums were used as raw materials to exclude from the analysis most of the triacylglycerols present in the oil, while preserving all other minor components that may be present in the "normal" matrix during degumming. Analysis of the gums by the P-31 NMR method revealed only phosphatidyl-containing compounds and did not reveal lyso-compounds or phosphorus compounds. Data on the phospholipid composition are listed below in Table 7. The content of diacylglycerol in the gums obtained by the technology of industrial aqueous degumming was 1.5 95.

Two control experiments were performed with reaction conditions optimal for each of the enzymes to determine baseline parameters for further analysis of the experiments. The first control test was carried out at a neutral pH for the enzyme phospholipase C, and the second test was carried out at a pH of 4.5, which is optimal for the enzyme phospholipase A.

Example 11

Control: Phospholipase C (PI C) at neutral pH - 50 g of raw soy gums were introduced into a round-bottom flask with a volume of 500 ml. 10 g of Rypipe "MM of the Megepisht company (RI C lipase, lot number 9980004A1) were added. The material was mixed with the help of a top-drive paddle stirrer equipped with a rounded blade made of stainless steel, fitted to the bend of the flask, at a speed of approximately 150 rpm. The opening of the flask was covered with a film Stir to avoid evaporation of water.The raw gums and enzyme were heated to 45°C with continuous stirring.

The system was maintained for eight hours. Then the installation was disassembled and the hydrolyzed gums were collected.

The gum was transferred to a centrifuge tube and centrifuged for 15 min at 5000 rpm to separate the light phase

Salt") from the heavy phase ("gum").

The content of Jude in the restored oil turned out to be equal to 32.6 95, in comparison with the initial content of SAC of 1.5 95, it differed by 31.1 95. A large increase in the content of OADS: natural in the reaction of RI C, in which OD does not enter into the reaction further. The phospholipid profile obtained by heavy phase analysis by the RZ1-NMR method confirmed that phosphatidyl groups were hydrolyzed to phosphate groups. Unexpectedly, a small amount of "I" was detected, just like small amounts of all lyso groups. Thus, RI C has some activity of RI A under the reaction conditions of this example.

Example 12

Control: Phospholipase A (PI A) at a pH of 4.5-50 g of raw soy gums was introduced into a 500 ml round-bottomed flask. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred for 5 min. Then 1.8 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for another 5 min. Citric acid and alkali formed a weak buffer with a pH of 4.5. Did you add 2 g of I esiaze? firm Momogutez (RI At lipase, batch number I ММО5007). The material was mixed using an overhead paddle stirrer equipped with a rounded stainless steel paddle fitted to the curve of the flask at a speed of approximately 150 rpm. The opening of the flask was covered with a RagayteU film to avoid water evaporation. Raw gums and enzyme were heated to 45 °C with continuous stirring.

The system was maintained for eight hours. Then the installation was disassembled and the hydrolyzed gums were collected.

The gum was transferred to a centrifuge tube and centrifuged for 15 min at 5000 rpm to separate the light phase

Salt") from the heavy phase ("gum").

The measured content of Jude in the reduced oil was 3.8 95, with an increase of 2.3 95. The phospholipid profile obtained by the analysis of the heavy phase by the R31-NMR method confirmed that the phosphatidyl groups were hydrolyzed to the corresponding lyso groups, which is natural in the reaction of RI A. Very small amounts of "C" and "E" were detected, just like "A".

RI A does not have significant activity of RI C.

Example 13

RIA at neutral pH - 50 g of raw soybean gums, separated with the help of an industrial centrifuge for degumming, were introduced into a round-bottom flask with a volume of 500 ml. Did you add 2 g of I esiaze? of the firm Momogutez (RI A lipase, batch number ГМО5007). The material was mixed using an overhead paddle stirrer equipped with a rounded stainless steel blade fitted to the curve of the flask at a speed of approximately 150 rpm.

Was the opening of the bulb covered with Ragate film? to avoid water evaporation. The raw gums and the enzyme were heated to 45"C with continuous stirring. The system was maintained for eight hours. Then the installation was disassembled and the hydrolyzed gums were collected. The gums were transferred to a centrifuge tube and centrifuged for 15 min at 5000 rpm to separate the light phase ("oil"). ) from the heavy phase ("gum"). The OAS content of the reconstituted oil was 2.6 95, with an increase of only 1.1 95 SDS. The phospholipid profile showed that all the "parent" gums were hydrolyzed, but a decrease in the amount of lyso - and phospho-derivatives, in comparison with the control conditions in example 12. The results suggest that under the reaction conditions of this example, the RI A enzyme does not form DA, oil or phosphorus compounds, but forms lyso-compounds and fatty acids.

Example 14

RIS and RIA at neutral pH - 50 g of raw soybean gums, separated using an industrial centrifuge for degumming, were introduced into a 500 ml round-bottomed flask. 10 g of Rigiipe"mm were added

Megepisht (RIS lipase, batch number 9080004A1) and 2 g of I esiabe? firm Momogutez (RI A1 lipase, batch no

ЮУМО5007). The material was stirred using a top-drive paddle stirrer equipped with a rounded stainless steel blade fitted to the curve of the flask at a speed of approximately 150 rpm. The opening of the flask was covered with a Ragaite film to avoid water evaporation. The raw gums and enzyme were heated to 45 °C with continuous stirring. The system was maintained for eight hours. Then the installation was disassembled and the hydrolyzed gums were collected. The gums were transferred to a centrifuge tube and centrifuged for 15 min at 5000 rpm to separate the light phase ("oil" ) from the heavy phase ("comedy").

The DA content in the recovered oil was only 7.8 90, compared to 32.6 95 As obtained when using

RIS as a single enzyme. The phospholipid profile confirmed that all phosphatidyl groups were hydrolyzed to lyso- and phosphate groups. Phosphate compounds "C", "E" and "A" were detected in approximately the same amounts as in example 11, except that the amount of "I" was significantly reduced. The amount of lyso-PE was slightly reduced, while the amount of lyso-RS and lyso-RA was approximately twice as high as in example 11, but did not increase much.

The amount of lyso-RI and lyso-RA was significantly higher than in example 11, because the enzyme RI A was also present in the "reaction matrix" and converted RI and RA to lyso-forms.

P-31 NMR analysis not only confirmed that RI C converted approximately the same amount of phospholipids to phosphate groups as in Control Example 11, but also confirmed that the remaining phosphatidyl groups were converted to lyso forms, indicating conversion by RI A. This result is surprising because the pH conditions were not optimal for the reaction involving RI A. The amount of SPAS present in the oil should have been about 33 95 and not 7.8 95 as determined by HPLC analysis.

Example 15

FIG. Her RIA at a pH of 4.5 - 50 g of raw soybean gums, separated with the help of an industrial centrifuge for degumming, were introduced into a round-bottom flask with a volume of 500 ml. Added 2.0 g of 50 95 wt./wt. citric acid solution and stirred for 5 min. Then 1.8 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for another 5 min. Citric acid and alkali formed a weak buffer with a pH of 4.5. 10 g of Rippe"M from the Megepitz company (RI C lipase, batch number Z?0VO004A1) and 2 g of I espase? from the Momogutez company (RI A1 lipase, batch number I UMO5007) were added. The material was mixed using a top-drive paddle mixer equipped with a rounded blade with of stainless steel, fitted to the bend of the flask, at a speed of about 150 rpm. The opening of the flask was covered with a film of Ragaite to avoid evaporation of water. The raw gums and enzyme were heated to 45 "C with continuous stirring.

The system was maintained for eight hours. Then the installation was disassembled and the hydrolyzed gums were collected.

The gum was transferred to a centrifuge tube and centrifuged for 15 min at 5000 rpm to separate the light phase

Salt") from the heavy phase ("gum").

The content of SAC in the reduced oil turned out to be the same as in example 14,7,8 906. The analysis of the phospholipid profile confirmed that all phosphatidyl groups were completely hydrolyzed to phosphate and lyso groups, as in examples 12-14. Phosphate groups, "C ", "E", "I" and "A" were detected in approximately the same quantities as in example 11. The content of lyso-RS and lyso-"PE was significantly higher than in example 11. As in example 13, amounts of lyso-RI and lyso-RA were significantly higher than in example 11, because the enzyme RI A was also present in the "reaction matrix" and converted RI and RA to lyso-forms. As in degumming examples 1-10, the amount of DO , actually found was smaller than expected based on the P-31 NMR analysis data, suggesting that the BAS and EPA formed were used in the subsequent TA formation reaction.

P-31 NMR analysis not only confirmed that RI C converted approximately the same amount of phospholipids to phosphate groups as in Control Example 11, but also confirmed that the remaining phosphatidyl groups were converted to their lyso forms by the enzyme RI A. The amount of SAC present in the oil should have been about 33 95, not 7.8 95 as determined by HPLC analysis.

Table 7 summarizes the phospholipid profiles obtained by the RZI NMR method, where all numbers express values in percent by weight of the heavy phases separated from the reaction mixtures of examples 11-15, showing the unreacted residues of phosphatidyl, lyso groups, which are formed in the reaction with the participation of RIA, and phosphate groups obtained in the reaction with RI C.

Table 7

Example | RS | RE | RI | RA | SRS | RE | GAME | RA | "C" | "E' | "| And yayu, zve zve |5ho| ее овгоо| oso oso 005 oso 000 ooo. substance 11 | 06001000 545)| 0.54 | 059 | 030 | 158 | 165 427 | 320 | 040 | 156 712 | 0001000 | 000 | 000 /19.91|1901| 1319/1080) 0.05 / 005 | 000 | 0.39 13 | 0oo|o000| 0001000 7.59 | 717 | 507 | 3.97 | 012 | 0.05 | 0.00 | 017 14 | 0о0|000| 000000 123 | 023 | 739 | 370 | 440 | 340 | 022 | 147,715 |000|000|000)| 000) 1.09 | 271 | 829 | 486 | 425 | 300 | 0.50 | 1.97

Table 8 summarizes the initial amounts of OASI and the acidity (AM) of the solutions of the original gums for each of the above-described examples 11-15, the theoretical amount of CAC and EPA that should have been present in the resulting oil if all the phospholipids in the original oil had reacted with enzymes, and actual amounts of SAC,

present in the resulting oil. The final RIB content was not measured because the EPA measurement procedure requires more oil than was available from the experimental data. In each of these examples, less RAS was detected in the recovered oil than expected theoretically, further supporting the conclusion that SDS is involved in the formation of TAC during the reaction of ODS with EPA.

Table 8 1111110 | Initial lecithin, | Enzyme | Theoretical output Restored oil o o o o o o 11711115 | 21 | FIG 402 | 00 | 326 | - 12 | 15 | 21 | RA | 175 | 241 | 38 | - 13 | 15 | 21 | REA | 15 | 241 | 26 | - ( 714 | 15 | 21 | RISRIA | 402 | 76 | 78 | - ( | 15 | 21 | RISRIA | 402 | 76 | 78 | - / x The theoretical amount of ODO includes only the amount formed from phosphatidylcholine and phosphatidylethanolamine. "x The theoretical amount of EPA includes the amount formed during the reaction of all phospholipids with RI A.

If RI C and RGA participate in the reaction together, then only EPA from phosphatidylserine, phosphatidylinositol, and phosphatidic acid is taken into account. Acidity (AM) is the number of milligrams of potassium hydroxide required to neutralize acids in one gram of sample (1). AM is a titrated acidity caused by phospholipids and free fatty acids. (2). The fraction of EPA was not measured due to insufficient amount of sample for titration.

The following example 16 is identical in procedural steps to example 15, except that the example is performed on a doubled scale. The purpose of this example was to summarize the material balance of the entire sample before and after the enzymatic reaction with the participation of RIA/РІС in order to confirm the fact of the formation of triacylglycerols from the side products of the reaction of hydrolysis of phospholipids by РА/РІС enzymes.

Example 16

RIS and RIA at a pH of 4.5 - 100 g of raw soybean gums, separated using an industrial centrifuge for degumming, were introduced into a round-bottom flask with a volume of 500 ml. Added 4.0 g of 50 95 wt./wt. citric acid solution and stirred for 5 min. Then 3.6 ml of a 4-molar sodium hydroxide solution was added and the mixture was stirred for another 5 min. Citric acid and alkali formed a weak buffer with a pH of 4.5. 20 g of Ripipe"M from the Megepitz company (RI C lipase, batch number 9?080004A1) and 4 g of I esiaze? from the Momogutez company (RI A1 lipase, batch number I KhMO5007) were added. The material was mixed using a top-drive paddle mixer equipped with a rounded blade with of stainless steel, fitted to the bend of the flask, at a speed of about 150 rpm. The opening of the flask was covered with a film of RagaiteU to avoid evaporation of water. The raw gums and enzyme were heated to 45 "C with continuous stirring.

The system was maintained for eight hours. Samples of the original gums and the enzymatically treated mixture were examined "as is" for moisture content, percent gum content and neutral oil content. The content of neutral oils was measured according to the method described below in the Appendix. Then, the separated neutral oils were analyzed for diacylglycerol content. The results are shown in Table 9.

Table 9 11111111 | Weekendcomedy8 | Enzymatically processed gums

The result of the analysis of raw raw gums is typical for raw gums obtained in the process of industrial aqueous degumming of soybean oil. The gum was about 73 95, and the neutral oil about 27 Uo, of the dry matter of the sample. The analysis of "enzymatically treated gums" in example 16 shows that a significant part of the phospholipids present was hydrolyzed by RI A/RI C enzymes, as indicated by a decrease in the content of gums from 73 to 41 95, while the amount of triacylglycerols increased from 26.5 95 to 58, 6 95. Theoretically, the expected amount of diacylglycerols formed in this process should have been 40.2 956, but it turned out to be only 13.2 95. It was concluded that the combination of RI C and RI A enzymes, used for oil degumming, cleaning or modification of lecithins forms triacylglycerols, or oil.

A method was described for the formation of triacylglycerols from phosphatidyl-containing oil gums by treating the gums with a combination of RHA and RI C enzymes, according to which CAC, a product of the RI C reaction, and EPA, a side product of the reaction

RIA, combine with each other in the presence of enzymes, forming TAI molecules. These two different enzymes can interact with the gums simultaneously or sequentially; in the case of a sequential method, the enzymes can be added in any order. The reaction time of the enzymes with the gums can be on the order of four hours or less and can be as short as about 30 min. Enzymes having RI A activity can be selected from the group consisting of phospholipase AT enzyme and phospholipase A2 enzyme. The RIA enzyme can be present in the reaction mixture at a concentration of approximately 2 ppm. of active enzyme or less; or 1 m.h. of active enzyme or less; or even as small as 0.5 m.h. of active enzyme or less. Enzymes having RIS activity can be selected from the group consisting of the enzyme phospholipase C and the enzyme phosphatidylinositol-specific phospholipase C. The RIS enzyme can be present in the reaction mixture at a concentration of about 30 ppm. of active enzyme or less; or 20 m.ch. of active enzyme or less; or even as small as 10 m.h. of active enzyme or less.

Enzymatic reactions can be carried out at a temperature in the range of about 40-80 "C, preferably in the range of about 40-60 "C. pH can have a value in the range of about 3-7. The enzymatic reaction can be facilitated by stirring, preferably for 45 s or longer when the reaction is carried out on a laboratory scale.

It is expected that the time spent on stirring will be increased when moving to industrial scales, as should be known to those skilled in the art. Also, precipitation of phosphorus-containing substances with acetone will allow recovery of the obtained oil; a similar procedure is known in the art for the production of defatted lecithins.

Although the preferred embodiments of the invention have been set forth herein as known at the time of application, other embodiments incorporating the method of the invention will be apparent to those skilled in the art, and all such embodiments and their equivalents are defined as such , which fall within the scope of this application, which is contained in the claims of this document.

The following method was used to measure neutral oils in the examples of this application.

Definition

This method measures the total neutral oil found in raw gums, lyso-gums, or crude oil soapstock.

Range of application

Application in relation to gums, lyso-gums and soap stocks.

Link

Methods approved by the American Society of Petrochemists (A.O.0.5.):

A.O.0.5. method with 5-40

A.O.0.5. method Са 26-55

A.O.0.5. method yes 4-46

Equipment 1. Measuring cylinder - 100 ml 2. Measuring cylinder - 50 ml 3. Measuring cylinder - 25 ml 4. Disposable centrifuge tubes - 50 ml (polypropylene) 5. Separating funnel - 500 ml 6. Laboratory beaker - 500 ml 7. Laboratory beaker - 400 ml 8. Laboratory beaker - 250 ml 9. Glass stirring rods 10. Centrifuge 11. Desiccator 12. Steam bath 13. Oven - 10570

Reagents 1. Aqueous potassium hydroxide (KOH) - 14 9o by weight. 2. Sodium chloride (Masi) - pure for analysis. 3. Ethyl alcohol - allowed formulations of specially denatured alcohol 30 and ZA, 5095 by volume.

Mix 10 volumes of 95 95 alcohol and 9 volumes of distilled water. 4. Ethyl alcohol - allowed formulations of specially denatured alcohol 30 and ZA, 10 95 by volume.

Mix 2 volumes of 95 95 alcohol and 17 volumes of distilled water. 5. Petroleum ether - the degree of purity corresponds to the standard of the American Chemical Society (AC5). 6. Acetone - the degree of purity corresponds to the standard of the American Chemical Society (AC5). 7. Deionized or distilled water. 8. Nitrogen - clean and dry.

Methodology 1. Measure the 95% humidity of the sample immediately after the sample has settled. Note: AOS 26-55, temperature reduced to 105 "C due to foaming of soap samples at 130 "C. Time increased to 4 hours. 2. Mix the sample thoroughly and weigh immediately.

Q. Weigh 5 g (to the nearest 0.0001 g) of sample into a pre-weighed 50 mL disposable centrifuge tube. (Note: including lid and beaker (to keep the centrifuge in balance)). 4. Add 35 mL of cold acetone (keep in an ice bath) to the sample and mix very well with a glass rod. Break up the lecithin precipitate with a glass rod. Note: Acetone turns bright yellow. 5. Close the centrifuge tube. 6. Centrifuge the acetone for 5 minutes to separate the gums from the acetone. 7. Pour acetone into a 250 ml laboratory beaker. 8. Repeat steps 4 through 7 four times.

and. After the last extraction, extract the gum and transfer it to a pre-weighed tub for drying.

Wait for excess acetone to evaporate. p. Place the sample overnight in a drying cabinet with a draft at 105 76. p. Cool to room temperature in a desiccator and weigh the contents of the drying tub and measure. and. Calculate the percentage of gum in the original sample and on a dry matter basis. 9. Pour a layer of acetone into a 500 ml separatory funnel ("A"). 10. Add 50 ml of water to the separatory funnel. Stir. 11. Add 50 ml of petroleum ether (PE). Stir. 12. Add a bunch of mass (1/4 tablespoon of table salt) to the dividing funnel. Stir. 13. Wait for the two layers to separate. Extract the lower layer (acetone/water), including possible emulsion, and transfer to a new separatory funnel ("8-1"). DO NOT THROW THE BALL R.E. 14. Add 50 mL of petroleum ether (PE) to the acetone/aqueous layer from step 13. Mix. 15. Wait for the two layers to separate. Extract the lower layer (acetone/water), including possible emulsion, and transfer to a new separatory funnel ("B-2"). 16. Add a layer of petroleum ether to the original extract of R.E. from stage 13 (separating funnel "A"). 17. Repeat steps 14 to 16 twice. The acetone/aqueous layer may be added to the already used separatory funnel "B-1" above. After the last extraction is complete, you can discard the acetone/aqueous layer. 18. Add 100 ml of 50 95 ethanol to the separatory funnel containing R.E. Stir. 19. Add 10 ml of 14 95 KOH. Mix carefully. 20. Add 50 mL of 50 95 ethanol to the separatory funnel. Stir. 21. Wait for the layers to separate completely. Do not allow the layer of R.E. was in contact with the alcohol/con layer for longer than 30 min. DO NOT THROW THE BALL R.E. 22. Extract the alcohol/KOH layer and transfer it to a new separatory funnel. 23. Add 50 ml of R.E. into a separatory funnel containing a layer of alcohol/con. Stir. 24. Wait for the layers to separate. Collect the alcohol/KOH layer and transfer to a new separatory funnel. Add a layer

RE. to R.E. of step 21. 25. Repeat steps 23 and 24. 26. Add 25 ml of 10 95 alcohol to the separatory funnel containing the RE layers, shake thoroughly. Wait for the layers to separate. Remove the alcohol layer. Discard the alcohol layer. 27. Repeat step 26 twice. To the third "wash" (alcohol layer), add two drops of phenolphthalein to make sure that the layer is neutral. If the layer turns pink, repeat step 26. 28. Carefully transfer the layer of R.E. into a tared glass, which was previously dried and cooled in a desiccator. Evaporate R.E. in a steam bath under a weak stream of nitrogen. 29. As soon as R.E. removed, place the beaker in an oven for 30 min at 105 C. 30. Cool in a desiccator to room temperature and weigh. 31. Repeat the procedure until the weight stops changing. (Constant weight is achieved when the loss (or excess) by weight does not exceed 0.1 95 during periodic 30-minute drying).

Count

Neutral oil, 95 (as is) - weight of neutral oil/weight of sample x100

Neutral oil, 95 (dry weight) - (mass of neutral oil/mass of samples 100 95 moisture content) x100

Gums, 95 (as is) - weight of dry gums/weight of sample x100

Gums, 95 (dry weight) - (mass of dry gums/mass of samples (100 95 moisture) x100 o o

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Claims (27)

1. The method of obtaining triacylglycerols from oil gums, which includes (a) providing an oil composition containing a certain amount of oil gums, where the specified gums contain phospholipids, (B) separating the oil gums from the oil composition to obtain the first fraction, where the gum impurities are separated, and the second fraction containing the separated oil gums, (c) treating the second fraction with one or more enzymes having RI.A activity to produce free fatty acids, (a) treating the second fraction with one or more enzymes having RIS activity, for the formation of diacylglycerols, in such a way that the indicated fatty acids and the indicated diacylglycerols interact with each other in the presence of at least one of the indicated enzymes, forming triacylglycerols. 2. The method according to claim 1, where stages (c) 1 (j) are carried out almost simultaneously. 3. The method according to claim 1, where stage (c) is carried out before stage (4). 4. The method according to claim 1, where step (4) is performed before step (c). 5. The method according to any one of claims 1-4, where the duration of the reaction of the enzymes with the second fraction is no more than about four hours. 6. The method according to claim 5, where the duration of the reaction of the second fraction with enzymes is approximately 30 minutes. 7. The method according to any one of claims 1-6, where one or more enzymes with RI.A activity are selected from the group consisting of the enzyme phospholipase A 1 and the enzyme phospholipase A2. 8. The method according to any of claims 1-6, where one or more enzymes with RIS activity are selected from the group consisting of the phospholipase C 1 enzyme, the phosphatidylinositol-specific phospholipase C enzyme. 9. The method according to any of claims 1-8, where the reaction of enzymes with the second fraction is carried out at a pH of about 8 or less. 10. The method according to claim 9, where the reaction of enzymes with the second fraction is carried out at a pH of about 3-7. 11. The method according to any of claims 1-10, where the reaction of enzymes with the second fraction is carried out at a temperature of about 40-80 2С. 12. The method according to claim 11, where the reaction of enzymes with the second fraction is carried out at a temperature of about 40-60 7C. 13. The method according to any one of claims 1-12, wherein the oil composition includes crude oil. 14. The method according to any one of claims 1-13, where the step of separating the gums from the oil composition is carried out by a method selected from the group consisting of aqueous degumming, acid degumming, alkaline refining and 1 enzymatic degumming, with the exception of degumming using a combination of RIA 1 RI/S. 15. The method according to claim 14, wherein the step of separating the gums from the oil composition is carried out by alkaline refining and the pH of the separated gums is adjusted to about 8 or less before treatment with the indicated enzymes. 16. The method according to any one of claims 1-15, where the specified RIS enzyme is present in an amount of about 1000 mg of active enzyme or less. 17. The method according to claim 16, where the indicated RIS enzyme is present in an amount of about 20 ppm of active enzyme or less. 18. The method according to claim 17, where the specified RIS enzyme is present in an amount of about 10 ppm of active enzyme or less. 19. The method according to any of claims 1-18, where the specified enzyme RI A is present in an amount of about 2 ppm of active enzyme or less. 20. The method according to claim 19, where the indicated enzyme RI.A is present in an amount of about 1 ppm of active enzyme or less. 21. The method according to claim 20, where the indicated RIA enzyme is present in an amount of about 0.5 ppm of active enzyme or less. 22. The method according to any one of claims 1-21, where the mixture of the second fraction 1 of enzymes is first mixed. 23. The method of claim 22, wherein the duration of mixing is at least about 45 seconds. 2A. The method according to any one of claims 1-23, which includes the step of adding some amount of water. 25. The method according to claim 24, where some amount of water is at least about 1.5 90 wt. based on the total mixture. 26. The method according to claim 25, wherein some amount of water is at least about 3.0 90 wt. based on the total mixture. 27. The method according to claim 26, where some amount of water is at least about 4.5 90 wt. based on the total mixture.